To remain healthy, cells, in particular mammalian cells, need to maintain a balance between oxidizing and reducing conditions sometimes referred to as redox state/potential. One of the most important mechanisms used to set and preserve the redox state/potential occurs through maintaining the relative amounts of oxidized and reduced glutathione in the cell.
Glutathione is a peptide made from three amino acids: glutamic acid, cysteine and glycine. It is often present at a much higher concentration in the cell than other proteins and peptides. Glutathione can exist in multiple forms in a cell. The two forms most often considered to relate to the cell's redox state/potential are GSH and GSSG. GSH, the reduced form, occurs when glutathione is unbound to other molecules (Monostori et al. 2009. J. Chromatography B 877:3331-3346). GSSG, the oxidized form, occurs when a disulfide bond exists between two glutathione molecules. GSH is often considered a first line of defense against oxidative damage and can remove active species from the cell and form GSSG. The presence of GSSG in plasma is often an indication of stress management. Since GSH and GSSG are related to each other through a simple oxidation/reduction reaction, they establish a redox state/potential within the cell. Further, since GSH and GSSG in total typically make up the highest redox couple in the cell, the determination of the amount of GSH and GSSG in a cell is usually performed, and the ratio of GSH to GSSG reported as a measure of the redox potential of the cell. Changes in the ratio of GSH and GSSG is often used as a measurement of oxidative damage in the cell.
Therefore, there exists a need to accurately and rapidly determine the ratio of GSH to GSSG in cells. Numerous reports indicate that compounds such as acetaminophen, tamoxifen, isoniazid and amodiaquine dramatically reduce the ratio of GSH to GSSG leading to cell death by apoptosis or necrosis (Srivastava et al. 2010. Handb. Exp. Pharmacol 196:165-94). Since variations in the ratio of GSH to GSSG have been reported to be associated with cell death, there is a need to be able to accurately determine this ratio (Monostori et al. 2009).
While multiple methods exist for the measurement of GSH in a sample, the most commonly used is the combination of an enzyme, such as glutathione reductase with Ellman's reagent (Monostori et al. 2009) and chromatographic methods, e.g. HPLC methods (Monostori et al. 2009). These methods utilize several processing steps including acidification, protein removal by precipitation, neutralization, internal control addition and others before sample addition for glutathione measurement (Monostori et al. 2009). As GSH is easily converted to GSSG, such as by exposure to air, preserving the initial amounts of GSSG and GSH in a sample for an accurate ratio measurement is difficult using techniques that require many processing steps. Therefore, these methods often require the addition of internal standards to allow estimations to be made of the amount of material lost and oxidized during the processing steps.
Measurement of the GSSG level in samples is even more difficult using the above methods. Since GSSG is typically only a small fraction of the GSH in the sample, relatively small changes in the amount of GSSG in a sample can result in dramatic changes in the GSH/GSSG ratio. For example, if the actual GSH/GSSG molar ratio is 100 (from a cellular concentration of GSH of 10 mM and GSSG of 100 μM), and the level of GSH drops from 10 to 9.8 mM (a 2% change) with an increase in GSSG of 100 μM [since two moles of GSH are needed to generate a mole of GSSG], the GSSG level changes from 100 μM to 200 μM [a 100% change], and the ratio of GSH to GSSG changes from 100 to 49 [a two fold change]. Thus, accurate measurement of the amount of GSSG in the sample is essential for correctly determining the GSH/GSSG ratio.
Several methods for determination of GSSG have been reported. A number of these methods calculate the level of GSSG in a sample by initially measuring the level of GSH and then measuring the level of GSH after reducing all of the GSSG to GSH (Monostori et al. 2009). The level of GSSG is then estimated by subtracting the amount of GSH found in the initial measurement of GSH from that of the level of GSH following reduction of GSSG to GSH. This results in the level of GSSG being determined by calculating the difference between two relatively large numbers, both of which have some degree of variability, and therefore, are highly error prone.
Other methods for the measurement of GSSG require that GSH first be chemically modified in the sample to prevent it from giving a signal in the GSH measurement reaction. Then, the GSSG in the sample is reduced to GSH and, finally, the resulting GSH generated from GSSG is measured. While such a method might be accurate in theory, there is a need to remove or inactivate the material used to mask the GSH before reducing the GSSG to GSH. If this is not done, the GSH formed will immediately be modified to the form generated by the blocking agent, resulting in an underestimate of the level of GSSG in the sample. In such cases, alkylating agents such as N-ethylmalamide (NEM) are used to rapidly and irreversibly modify the GSH into a form that will not give signal in the GSH measurement reaction. Unfortunately, these methods rely on the use of chemical reactions that give signal with any sulfhydryl reagent present in the sample. Thus, the alkylating reagent usually cannot be simply exhausted by addition of an excess of a sulfhydryl reagent that will exhaust the material. For this reason, these methods must remove all traces of excess reagent (such as NEM) resulting in the need to extract the solution—up to 9 times—greatly complicating sample processing and increasing the possibility of GSH loss.
Therefore, there is a need for a method to determine the amount of GSH, GSSG and/or the ratio of GSH to GSSG in an accurate and rapid manner, specifically a method is needed that requires few, if any, processing steps. The method of the present invention uses an enzymatic reaction for the measurement of the amount of GSH in a sample which requires no processing steps and prevents GSH loss.